Bacillus cereus is one of the important food pathogens. Since B. cereus group cells, such as B. cereus, B. thuringiensis, B. anthracis and B. mycoides, share many phenotypical properties and a high level of chromosomal sequence similarity, it is interesting to investigate the virulence profiles for B. cereus group cells, including B. cereus strains isolated from foods and samples associated with food‐poisoning outbreaks. For this investigation, the presence of enterotoxin genes, such as those of haemolysin BL, B. cereus enterotoxin T and enterotoxin FM, were assayed by polymerase chain reaction (PCR) methods. Meanwhile, their enterotoxin activities were assayed using the BCET‐RPLA kit, haemolytic patterns on sheep blood agar and their cytotoxicity to Chinese hamster ovary (CHO) cells. Results showed that there were 12 enterotoxigenic profiles for the 98 B. cereus group strains collected. In addition, if any of the three types of enterotoxins was present in the B. cereus group cells, these cells were shown to be cytotoxic to the CHO cells. Similar enterotoxigenic profiles could be found among strains of B. cereus, B. mycoides and B. thuringiensis. Thus, all B. cereus group strains may be potentially toxigenic and the detection of these cells in foods is important. We thus designed PCR primers, termed Ph1/Ph2, from the sphingomyelinase gene of B. cereus cells. These primers were specific for all B. cereus group strains and could be used for the detection of B. cereus cells contaminated in food samples.

Hemolysin BL (HBL) is a major virulence factor for Bacillus cereus group strains. It is also a target enterotoxin for the most commonly used B. cereus detection kit, i.e., the B. cereus enterotoxin (diarrheal type) reversed passive latex agglutination (BCET-RPLA) test kit. A survey of the HBL activities and the cytotoxicities to the Chinese hamster ovary (CHO) cells for the B. cereus group strains, however, showed that although only part of the B. cereus group strains are HBL active, all strains show cytotoxicity to the CHO cells. Thus, methods that allow the detection of not only the HBL but also of the B. cereus group strains are important. In this study, by comparison of the gene sequences of the 16S rRNA for B. cereus group and other bacteria strains, we designed primers B16S1 and B16S2 specific to all the B. cereus group strains. In addition, because HBL is a major enterotoxin, we also designed HBL gene-specific polymerase chain reaction (PCR) primers, i.e., Hm1 and Hm2, that generated the same results as those of the hemolysis and BCET-RPLA assays. Primers B16S1/B16S2 and Hm1/Hm2 could be combined into a multiplex PCR system for the simultaneous detection of B. cereus group cells and the possible presence of their HBL enterotoxins. Also, all these PCR systems allowed the detection of n x 10(0) CFU B. cereus cells per g of food sample if an 8-h enrichment step was performed prior to the PCR.

We investigated the bactericidal activity and exclusion effect of 10 strains of lactic acid bacteria (LAB) isolated from different commercial food products and infant feces against Helicobacter pylori (H. pylori) in human gastric epithelial AGS cells. Antagonistic activity of spent culture supernatants (SCS) from LAB (LAB-SCS) was tested, and the content of organic acids in SCS was analyzed with high-performance liquid chromatography (HPLC). In addition, the bactericidal activities of LAB-SCS were estimated by a time-kill assay and by measuring the exclusion effect of LAB-SCS against H. pylori in AGS cells. The results showed that SCS from certain strains with higher concentrations of organic acids dramatically decreased the viability of H. pylori. We also proved that the organic acids could inhibit H. pylori adhesion and invasion of AGS cells. Furthermore, the concentration and speciation of organic acids in SCS after fermentation of LAB are important factors in the inhibition of H. pylori infection. In addition, the in vitro methods used in this study might provide for the rapid screening of potential probiotics with anti-H. pylori activity in the dairy industry.

PCR primers specific for the detection of Lactobacillus acidophilus, Lactobacillus casei group, Lactobacillus delbrueckii, and Bifidobacterium longum were designed based on the elongation factor Tu gene (tuf). The specificity of these four primer sets were confirmed by PCR with 88 bacterial strains of Lactobacillus, Enterococcus, Bifidobacterium, and other bacterial species. Results indicated that these primer sets generated predicted PCR products of 397, 230, 202, and 161 bp for L. acidophilus, L. delbrueckii, L. casei group, and B. longum, respectively. Bacterial species other than the target organisms tested did not generate false-positive results. When these four primer sets were combined for the simultaneous detection of the lactic acid bacteria (LAB) in fermented milk products including yogurt, the LAB species listed on the labels of these products could be identified without the preenrichment step. The identification limit for each LAB strain with this multiplex PCR method was N X 10(3) CFU/ml in milk samples. The results of our multiplex PCR method were confirmed by PCR assay using primers based on the 16S rDNA or the 16S-23S intergenic spacer region and by biochemical tests using the API 50 CHL kit. When this multiplex PCR method was used with the determination of counts of total viable LAB and bifidobacteria, the quality of commercial fermented milk products could be assured.

Due to the increasing use of bifidobacteria in probiotic products, it is essential to establish a rapid method for the qualitative and quantitative assay of the bifidobacteria in commercial products. In this study, partial sequences of the tuf gene for 18 Bifidobacterium strains belonging to 14 species were determined. Alignment of these sequences showed that the similarities among these Bifidobacterium species were 82.24% to 99.72%. Based on these tuf gene sequences, 6 primer sets were designed for the polymerase chain reaction (PCR) assay of B. animalis subsp. animalis, B. animalis subsp. lactis, B. bifidum, B. breve, B. longum subsp. infantis, B. longum subsp. longum, and the genus of Bifidobacterium, respectively. These Bifidobacterium species are common probiotic species present in dairy and probiotic products. When each target Bifidobacterium spp. was assayed with the designed primers, PCR product with expected size was generated. In addition, for each target species, more than 70 bacterial strains other than the target species, including strains of other Bifidobacterium species, strains of Lactobacillus spp., Enterococcus spp., and other bacterial species, all generated negative results. PCR assay with primers specific to B. animalis subsp. lactis and B. longum subsp. longum confirmed the presence of these Bifidobacterium species in commercial yogurt products. In addition, for each product, enumeration of the bifidobacteria cells by culture method with BIM-25 agar and the quantitative real-time PCR showed similar cell counts. Such results indicated that within 15-d storage (4 °C) after manufacture, all the bifidobacteria cells originally present in yogurt products were viable and culturable during the storage.

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